Developing Integrated Approaches for Testing and Assessment (IATAs) in order to support nanomaterial safety.

Due to the unique characteristics of nanomaterials (NM) there has been an increase in their use in nanomedicines and innovative medical devices (MD). Although large numbers of NMs have now been developed, comprehensive safety investigations are still lacking. Current gaps in understanding the potential mechanisms of NM-induced toxicity can make it challenging to determine the safety testing necessary to support inclusion of NMs in MD applications. This article provides guidance for implementation of pre-clinical tailored safety assessment strategies with the aim to increase the translation of NMs from bench development to clinical use. Integrated Approaches to Testing and Assessment (IATAs) are a key tool in developing these strategies. IATAs follow an iterative approach to answer a defined question in a specific regulatory context to guide the gathering of relevant information for safety assessment, including existing experimental data, integrated with in silico model predictions where available and appropriate, and/or experimental procedures and protocols for generating new data to fill gaps. This allows NM developers to work toward current guidelines and regulations, while taking NM specific considerations into account. Here, an example IATA for NMs with potential for direct blood contact was developed for the assessment of haemocompatibility. This example IATA brings together the current guidelines for NM safety assessment within a framework that can be used to guide information and data gathering for the safety assessment of intravenously injected NMs. Additionally, the decision framework underpinning this IATA has the potential to be adapted to other testing needs and regulatory contexts.

Measuring particle size distribution and mass concentration of nanoplastics and microplastics: addressing some analytical challenges in the sub-micron size range.

HYPOTHESIS: The implementation of the proposal from the European Chemical Agency (ECHA) to restrict the use of nanoplastics (NP) and microplastics (MP) in consumer products will require reliable methods to perform size and mass-based concentration measurements. Analytical challenges arise at the nanometre to micrometre interface, e.g., 800 nm-10 µm, where techniques applicable at the nanometre scale reach their upper limit of applicability and approaches applicable at the micrometre scale must be pushed to their lower limits of detection. EXPERIMENTS: Herein, we compared the performances of nine analytical techniques by measuring the particle size distribution and mass-based concentration of polystyrene mixtures containing both nano and microparticles, with the educational aim to underline applicability and limitations of each technique. FINDINGS: Light scattering-based measurements do not have the resolution to distinguish multiple populations in polydisperse samples. Nanoparticle tracking analysis (NTA), nano-flowcytometry (nFCM) and asymmetric flow field flow fractionation hyphenated with multiangle light scattering (AF4-MALS) cannot measure particles in the micrometre range. Static light scattering (SLS) is not able to accurately detect particles below 200 nm, and similarly to transmission electron microscopy (TEM) and flow cytometry (FCM), is not suitable for accurate mass-based concentration measurements. Alternatives for high-resolution sizing and concentration measurements in the size range between 60 nm and 5 µm are tunable resistive pulse sensing (TRPS) and centrifugal liquid sedimentation (CLS), that can bridge the gap between the nanometre and micrometre range.

Asymmetric-flow field-flow fractionation for measuring particle size, drug loading and (in)stability of nanopharmaceuticals. The joint view of European Union Nanomedicine Characterization Laboratory and National Cancer Institute - Nanotechnology Characterization Laboratory.

Asymmetric-flow field-flow fractionation (AF4) has been recognized as an invaluable tool for the characterisation of particle size, polydispersity, drug loading and stability of nanopharmaceuticals. However, the application of robust and high quality standard operating procedures (SOPs) is critical for accurate measurements, especially as these complex drug nanoformulations are most often inherently polydisperse. In this review we describe a unique international collaboration that lead to the development of a robust SOP for the measurement of physical-chemical properties of nanopharmaceuticals by multi-detector AF4 (MD-AF4) involving two state of the art infrastructures in the field of nanomedicine, the European Union Nanomedicine Characterization Laboratory (EUNCL) and the National Cancer Institute-Nanotechnology Characterisation Laboratory (NCI-NCL). We present examples of how MD-AF4 has been used for the analysis of key quality attributes, such as particle size, shape, drug loading and stability of complex nanomedicine formulations. The results highlight that MD-AF4 is a very versatile analytical technique to obtain critical information on a material particle size distribution, polydispersity and qualitative information on drug loading. The ability to conduct analysis in complex physiological matrices is an additional very important advantage of MD-AF4 over many other analytical techniques used in the field for stability studies. Overall, the joint NCI-NCL/EUNCL experience demonstrates the ability to implement a powerful and highly complex analytical technique such as MD-AF4 to the demanding quality standards set by the regulatory authorities for the pre-clinical safety characterization of nanomedicines.

Multiplex profiling identifies clinically relevant signalling proteins in an isogenic prostate cancer model of radioresistance.

The exact biological mechanism governing the radioresistant phenotype of prostate tumours at a high risk of recurrence despite the delivery of advanced radiotherapy protocols remains unclear. This study analysed the protein expression profiles of a previously generated isogenic 22Rv1 prostate cancer model of radioresistance using DigiWest multiplex protein profiling for a selection of 90 signalling proteins. Comparative analysis of the profiles identified a substantial change in the expression of 43 proteins. Differential PARP-1, AR, p53, Notch-3 and YB-1 protein levels were independently validated using Western Blotting. Pharmacological targeting of these proteins was associated with a mild but significant radiosensitisation effect at 4Gy. This study supports the clinical relevance of isogenic in vitro models of radioresistance and clarifies the molecular radiation response of prostate cancer cells.

Measuring particle size distribution of nanoparticle enabled medicinal products, the joint view of EUNCL and NCI-NCL. A step by step approach combining orthogonal measurements with increasing complexity.

The particle size distribution (PSD) and the stability of nanoparticles enabled medicinal products (NEP) in complex biological environments are key attributes to assess their quality, safety and efficacy. Despite its low resolution, dynamic light scattering (DLS) is the most common sizing technique since the onset of NEP in pharmaceutical technologies. Considering the limitations of the existing sizing measurements and the challenges posed by complex NEPs both scientists and regulators encourage the combination of multiple orthogonal high-resolution approaches to shed light in the NEP sizing space (e.g. dynamic light scattering, electron microscopy, field flow fractionation coupled to online sizing detectors, centrifugal techniques, particle tracking analysis and tunable resistive pulse sensing). The pharmaceutical and biotechnology developers are now challenged to find their own pragmatic characterisation approaches, which should be fit for purpose and minimize costs at the same time, in a complicated landscape where only a few standards exist. In order to support the community, the European Nanomedicine Characterisation Laboratory (EUNCL) and the US National Cancer Institute Nanotechnology Characterization Laboratory (NCI-NCL) have jointly developed multiple standard operating procedures (SOPs) for NEP assessment, including the measurements of particle size distribution, and are offering wide access to their 'state of the art' characterisation platforms, in addition to making SOPs publicly available. This joint perspective article would like to present the NCI-NCL and EUNCL multi-step approach of incremental complexity to measure particle size distribution and size stability of NEPs, consisting of a quick preliminary step to assess sample integrity and stability by low resolution techniques (pre-screening), followed by the combination of complementary high resolution sizing measurements performed both in simple buffers and in complex biological media. Test cases are presented to demonstrate: i) the need for employing at least one high-resolution sizing technique, ii) the importance of selecting the correct sizing techniques for the purpose, and iii) the robustness of utilizing orthogonal sizing techniques to study the physical properties of complex NEP samples.

Enantioselective cytotoxicity of ZnS:Mn quantum dots in A549 cells.

Chirality strongly influences many biological properties of materials, such as cell accumulation, enzymatic activity, and toxicity. In the past decade, it has been shown that quantum dots (QDs), fluorescent semiconductor nanoparticles with unique optical properties, can demonstrate optical activity due to chiral ligands bound on their surface. Optically active QDs could find potential applications in biomedical research, therapy, and diagnostics. Consequently, it is very important to investigate the interaction of QDs capped with chiral ligands with living cells. The aim of our study was to investigate the influence of the induced chirality of Mn-doped ZnS QDs on the viability of A549 cells. These QDs were stabilized with D- and L-cysteine using a ligand exchange technique. The optical properties of QDs were studied using UV-Vis, photoluminescence (PL), and circular dichroism (CD) spectroscopy. The cytotoxicity of QDs was investigated by high content screening analysis. It was found that QDs stabilized by opposite ligand enantiomers, had identical PL and UV-Vis spectra and mirror-imaged CD spectra, but displayed different cytotoxicity: QDs capped with D-cysteine had greater cytotoxicity than L-cysteine capped QDs.

Characterization of SH-SY5Y human neuroblastoma cell growth over glass and SU-8 substrates.

The physical properties of substrates can have profound effects on the structure and function of cultured cells. In this study, we aimed to examine the viability, adherence, and morphological and functional variations between SH-SY5Y human neuroblastoma cells cultured on SU-8 surfaces compared with control surfaces composed of borosilicate glass, which are routinely used for cell culture. The SU-8 polymer has been extensively studied for its biocompatibility, but there has been little investigation into the characteristic differences between cells cultured on SU-8 when compared with glass. SH-SY5Y cells were cultured within polydimethylsiloxane wells on both SU-8 and glass substrates for up to 72 h after which flow cytometry and enzyme-linked immunosorbent assay analysis was performed to examine cell viability and neurotoxicity. Immunocytochemistry was also performed to analyze the morphological and functional characteristics of the cells. Atomic force microscopy was performed to measure surface roughness and to map cell-substrate interactions. Nanoindentation testing was used to characterize the mechanical properties of polymer surface. Results showed that SH-SY5Y cells grown on SU-8 have significantly improved viability and increased morphological and functional characteristics of neurodevelopment. The results from this study suggest that the mechanical properties of the polymer are optimal for the study of cultured cell lines, which could account for the increased viability, adherence, and morphological and functional characteristics of neurodevelopment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2129-2138, 2017.

Nanotechnologies for the study of the central nervous system.

The impact of central nervous system (CNS) disorders on the human population is significant, contributing almost €800 billion in annual European healthcare costs. These disorders not only have a disabling social impact but also a crippling economic drain on resources. Developing novel therapeutic strategies for these disorders requires a better understanding of events that underlie mechanisms of neural circuit physiology. Studying the relationship between genetic expression, synapse development and circuit physiology in CNS function is a challenging task, involving simultaneous analysis of multiple parameters and the convergence of several disciplines and technological approaches. However, current gold-standard techniques used to study the CNS have limitations that pose unique challenges to furthering our understanding of functional CNS development. The recent advancement in nanotechnologies for biomedical applications has seen the emergence of nanoscience as a key enabling technology for delivering a translational bridge between basic and clinical research. In particular, the development of neuroimaging and electrophysiology tools to identify the aetiology and progression of CNS disorders have led to new insights in our understanding of CNS physiology and the development of novel diagnostic modalities for therapeutic intervention. This review focuses on the latest applications of these nanotechnologies for investigating CNS function and the improved diagnosis of CNS disorders.

Influence of strong static magnetic fields on primary cortical neurons.

Intense uniform magnetic fields, such as those used in magnetic resonance imaging (MRI), are thought to exert little influence at the cellular level. Here we report modifications of the signaling cascades in rat cortical neurons cultured for 1 h in magnetic fields of up to 5 Tesla. The activation of c-Jun N-terminal kinase (JNK) increases monotonically with field strength, with a maximal activation of approximately 10% at 5 T, whereas the activation of extra cellular-regulated kinase (ERK) shows a maximum at 0.75 T ( approximately 10%). Since ERK is involved in cellular differentiation, these results indicate a magnetic induction of the signaling events associated with differentiation. However, the cells respond to further increases in field strength by evoking a stress response, since JNK is a stress-activated protein kinase. Three possible mechanisms are discussed and of these, the most plausible is magnetic field induced change in the membrane rest potential, a microscale magnetohydrodynamic effect. This mechanism most likely involves the activation of voltage dependent Ca(2+) channel opening; since intracellular Ca(2+) concentration was also found to be modified by the static magnetic field.

Endothelial cell alignment on cyclically-stretched silicone surfaces.

Endothelial cells at the interface between the bloodstream and the vessel wall are continuously subjected to mechanical stimulation in vivo, and it widely recognised that such stimulation plays an important role in cardiovascular physiology. Cell deformation is induced by mechanical forces such as cyclic stretch, fluid shear stress, and transmural pressure. Although much of the work in this field has dealt with the effect of fluid shear stress, very little is known about how cyclic forces modulate and alter the morphology of single endothelial cells, and thereafter, how they effect the confluent layer of endothelial cells lining the vessel wall. The aim of this study is to investigate the response of endothelial cells when subjected to substrate deformation of similar magnitude to those experienced in vivo. Human umbilical vein endothelial cells (HUVEC) were cultured on plasma-treated silicone strips and uni-axially cyclically stretched using a custom made mechanical device. Results showed that endothelial cells subject to 10% deformation for as little as 4 h reoriented perpendicular to the stretch direction. In addition, although no integrin coating was applied to the substrate, it was found that plasma-treated silicone provided a cell adhesion substrate comparable to the commonly used collagen type I. Thus the results show that the stretch stimulus alone affects the morphology of endothelial cells. Further studies are required to establish the relative importance of substrate strain vs. fluid flow stimuli.

Compression data on bovine bone confirms that a "stressed volume" principle explains the variability of fatigue strength results.

The literature contains many measurements of the fatigue properties of compact bone, but these experimental results have been difficult to interpret and use due to a large amount of apparent scatter: variation in the number of cycles to failure for a given cyclic stress or strain range. Recently Taylor (1998a, Journal of Orthopaedic Research, 16, 163-169) showed that much of this scatter could be explained using a statistical model which took into account specimen size, or more specifically stressed volume. The present paper describes an attempt to test this model by using it to predict some new data, for bovine bone tested in compressive loading at room temperature at physiological loading rates. Twenty specimens were tested at the same applied load range (100 MPa). The theory was able to predict the mean behaviour of the specimens very well, with an accuracy (expressed in terms of stress) of 2%. It was also able to predict the degree of scatter (i.e. the variation of Nf), which was shown to be similar to that measured by other workers.